Tryptophan is one of essential amino acids, and has been broadly used in diverse fields including feed additives, medical substances such as sleeping draught or tranquilizer or Ringer's solution, and health food substances. Typical production methods of Tryptophan are chemical synthesis, enzyme reaction, and fermentation using microorganisms. In case of the chemical synthesis, the production takes place in a high temperature and high pressure space, and because D-tryptophan and L-tryptophan are produced together an additional refining process is required to obtain desired tryptophan. In case of the enzyme reaction such as the Japanese patent to Matsui Doatsui (Korean Patent Publication No. 90-005773), indole and serine used as substrates for the reaction are very expensive and the enzyme itself is not safe.
On the other hand, the fermentation using microorganisms involve auxotrophic strains and regulatory mutant strains of diverse microorganisms such as E. coli and Corynebacterium. Rapid technical advances in gene recombination in 1980's have provided much information on metabolism and control mechanism thereof. Many researchers had remarkable successes to develop superior recombinant strains through gene manipulation, and to improve productivity (Matsui et al, 1988). Also, in Korea, a number of Tryptophan production techniques related with the direct fermentation were disclosed either by using Tryptophan-resistant or auxotrophic mutant strains (Korean Patent Publication Nos. 87-1813, 90-8251, and 92-7405) or recombined strains (Korean Patent Publication Nos. 90-5772 and 91-5627). Mainly these Tryptophan analog resistant strains were to overcome feedback inhibition of enzymes during the Tryptophan biosynthesis, and the recombinant strains were also used for cloning enzymes during the Tryptophan biosynthesis. In fact, the studies have made a remarkable success. For instance, the biggest merit of the traditional L-tryptophan production using an artificial mutant of E. coli was that inexpensive cultivation substrates were used to product the L-tryptophan. However, the productivity or the Tryptophan yield was extremely low. Therefore, to maximize the Tryptophan yield through the gene recombination, there exists a need to secure an artificial mutant which is excellent as a parent strain and obtain genes whose regulations are released.
Therefore, the present invention has been made in view of the above problems, and it is an object of the present invention to identify base sequences and amino acid sequences of a mutant gene for encoding aroF and aroG, which are enzymes for use in the synthesis of 3-deoxyarabionohep-tulosonate 7-phosphate (hereinafter it will be referred to as DAHP), the first precursor of aromatic amino acids during the biosynthesis of Tryptophan originated from an E. coli mutant strain CJ285, out of phosphoenolpyruvate and Erythorse 4-phosphate, and trpR and tyrR for regulation transcription of genes related with the Tryptophan synthesis.
It is another object of the present invention to provide an L-tryptophan producing E. coli mutant strain that contains single or multi mutant genes described above.
It is still another object of the present invention to provide a production method of L-tryptophan with high concentration and high yield by cultivating the mutant directly in a fermentation medium containing glucose.